Comparison of Conventional Methods, MALDI-TOF MS and Sequence Analysis Methods for Identification of Aspergillus Species Isolated from Clinical Samples

Amaç: Aspergillus tu?ru? mantar enfeksiyonları immu?n sistemi baskılanmış hastalarda, yu?ksek mortalite ve morbidite ilesonuçlanan invazif hastalıklara yol açmaktadır. Bu nedenle hızlı ve dog?ru tanı konularak uygun antifungal tedavibaşlanması invazif aspergillozlu hastalar için hayati o?neme sahiptir. Gu?nu?mu?zde daha hızlı, kolay uygulanabilir, yu?ksekduyarlılık ve o?zgu?llu?g?e sahip yeni tanı yo?ntemleri tercih edilmektedir. Bu çalışmada çeşitli klinik o?rneklerden izoleedilen Aspergillus tu?rlerinin; geleneksel yo?ntemler, MALDI-TOF MS sistemi ve DNA dizi analizi yo?ntemi kullanılaraktanımlanması ve bu yo?ntemlerin karşılaştırılması amaçlanmıştır.Yo?ntemler: Bu çalışmada çeşitli klinik o?rneklerden izole edilen toplam 50 Aspergillus izolatı çalışmaya dahil edildi.Aspergillus suşlarından 2 tanesi kontaminasyondan dolayı çalışma dışı bırakıldı.Bulgular: Çalışmamızda referans tanımlama yo?ntemi olarak kullandıg?ımız ITS bo?lgesinin dizi analiziyle, suşların 25tanesi A.fumigatus tu?r kompleksi (%52,08), 17’si A.flavus tu?r kompleksi (%35,42), 3’u?A.niger tu?r kompleksi (%6,25),2’si A.terreus tu?r kompleksi (%4,17), 1’i A.sydowii tu?r kompleksi (%2,08) olarak tanımlandı. Altın standart yo?ntemindizi analizi olduğu ve geleneksel yöntem ile karşılaştırıldığında %97,9 uyum olduğu gözlendi. İki farklı yazılımkullandığımız MALDI-TOF MS sisteminde ise güncel IVD (invitro diagnostik) VITEK MS V.2.0 yazılımı ile doğrutanımlanan köken 37(%77,1) iken SARAMIS 4.12 RUO yazılımı ile doğru tanımlanan köken 42(%87,5) olarakbulundu.Sonuç: Moleküler yöntemler, geleneksel yöntemlerin yetersiz kaldığı ve tür tanımının yapılamadığı durumlardatamamlayıcı yöntem olarak kullanılabilir. Zaman açısından değerlendirildiğinde MALDI-TOF yöntemi hızlı ve duyarlıbir yöntem olmasına rağmen veri tabanının geliştirilmesi amacıyla suş sayısının arttırılarak bu tür çalışmalarıntekrarlanması gerekir.Objective: Fungal infections associated with Aspergillus species cause invasive diseases leading high mortality and morbidity as a result of deficiency of primary defence systems in immunosuppressive patients. Therefore, starting appropriate antifungal treatment after rapid and accurate diagnosis is critically important for invasive aspergillosis patients. Nowadays, more rapid, easily applicable, having high sensitivity and specifity new diagnostic methods are required. In this study, identification of Aspergillus spp. isolated from various clinical samples by using conventional methods, MALDI-TOF MS system and DNA sequence analysis and comparison of the methods are aimed. Methods: Totally 50 Aspergillus strains were included in this study. Two isolates were excluded from the study due to contamination. Results: 25 strains were identified as A.fumigatus species complex (52.08%), 17 strains were identified as A.flavus species complex (35.42%), 3 strains were identified as A.niger species complex (6.25%), 2 strains were A.terreus species complex (4.17%) and 1 strain was A.sydowii species complex (2.08%) byITS sequence analysis method used as reference diagnostic method in this study. The sequence analysis wasthe gold standard method and it was observed that there was 97.9% compliance compared to the conventional method. Two different software programmes were used for MALDI-TOF MS system. 37 strains (77.1%) were accurately defined by current IVD (in vitro diagnostic) VITEK MS V.2.0 whereas 42 strains (87.5%) were accurately defined by SARAMIS 4.12 RUO software programme. Conclusions: Molecular methos are thought to be appropriate to be used as complementary method when conventional methods are insufficient for identification at the species level. Although MALDI-TOF MS method is rapid and sensitive method when evaluated in terms of time, it is concluded that such studies should be repeated with more strains to develop database

Burkholderia cepacia complex bacteremia outbreaks among non-cystic fibrosis patients in the pediatric unit of a university hospital

Background. Burkholderia cepacia complex (Bcc) comprises multi-drug resistant, Gram-negative, motile, and aerobic bacteria. Bcc causes severe nosocomial infections particularly in patients with intravascular catheters and in those with cystic fibrosis. We studied a Bcc outbreak in non-cystic fibrosis patients. Methods. We analyzed data from six patients hospitalized at our center. Blood cultures identified as infectious were incubated onto 5% blood sheep agar, chocolate agar, and eosin methylene blue (EMB) agar. We examined possible sites that could be sources of infection at the clinic. We confirmed isolations with pulsed-field gel electrophoresis (PFGE) tests. Results. The first patient was hospitalized due to left renal agenesis, urinary tract infection, and renal failure. Bcc was isolated in blood cultures obtained due to high fever on the third day of hospitalization. We stopped new patient hospitalizations after detecting Bcc in blood cultures of other five patients. We did not detect further positive specimens obtained from other clinic and the patient rooms. PFGE patterns were similar in all clinical isolates of Bcc indicating that the outbreak had originated from the source. Conclusions. Bcc infection should be considered in cases of nosocomial outbreaks of multi-drug resistant organisms that require hospitalization at intensive care units. Control measures should be taken for prevention of nosocomial infections and required investigations should be done to detect the source of infection

Tuberculous peritonitis in a case receiving continuous ambulatory peritoneal dialysis(CAPD) treatment

BACKGROUND: Tuberculosis continues to be an important health problem in the world. Besides pulmonary involvement extrapulmonary involvement becomes an affair in developing countries, even in developed countries. CASE PRESENTATION: A thirty-six year old male patient was admitted with abdominal pain, diarrhea, nausea, vomiting and fever which had started one week before. The patient had been followed up with predialisis Chronic Renal Failure(CRF) diagnosis for 4 years and receiving continuous ambulatory peritoneal dialysis (CAPD) treatment for 4 months. In peritoneal fluid, 1600/mm3 cells were detected and 70% of them were polymorphonuclear leukocytosis. The patient begun nonspesific antibiotherapy but no benefit was obtained after 12 days and peritoneal fluid bacterial cultures remained negative. Peritoneal smear was positive for Asid-fast basilli (AFB), and antituberculosis therapy was started with isoniazid, rifampicine, ethambutol and pyrazinamide. After 15 days his peritoneal fluid cell count was decreased and his symptoms were relieved. Peritoneal fluid tuberculosis culture was found positive. CONCLUSION: Considering this case, we think that in patients with CAPD catheter and peritonitis; when peritoneal fluid leukocytes are high and PMNL are dominant, AFB and tuberculosis culture must be investigated besides bacterial culture routinely

Superficial Fungal Infections in Patients with Hematologic Malignancies: A Case-Control Study

Background and Design: Dermatophytes, yeasts and some moulds settle on the skin and mucosal surfaces in immunocompetent individuals as commensals. Patients with diabetes mellitus, HIV-positive patients, organ transplant recipients and the patients with malignancies are predisposed to develop superficial fungal infections. We aimed to determine the prevalence, clinical and mycological features of superficial fungal infections in patients with hematologic malignancies in this case-control study.Material and Method: Eighty patients with hematologic malignancies (49 men, 31 women) and 50 healthy individuals (22 men, 28 women) randomly selected at our clinical department as controls were included to this study between 2003 and 2004. The mean age was 52±1.85 years in patients and 41.56±2.04 years in controls. All patients were inspected for superficial fungal infections. Skin scrapings and mucosal swabs were obtained from the toe web, inguinal region, any suspicious lesion and oral mucosa. Nail samples were also collected. All samples were examined by direct microscopy and cultured in Sabouraud dextrose agar (SDA). The yeasts were established in germ-tube production. Results: Fifty-six (70%) of 80 patients with hematologic malignancies had fungal colonization, whereas 21 (42%) of 50 controls had. For both groups, oral mucosa was the predominant area that fungus was mostly isolated from. A rising number of non-dermatophyte moulds (26%) was observed. Candida albicans was the predominant agent isolated from the culture.Conclusion: The prevalence of superficial fungal infection was higher in patients with hematologic malignancies (being immunosuppressed) than in the normal population. Candida albicans was the predominant isolated agent that was found in our study. We observed oral mucosa candidal infection mostly. The rising number of non-dermatophyte moulds is attributed to long-term use of antibiotics, cytotoxic chemotherapies and antifungals

COVID-19 HASTALARINDA SEKONDER ENFEKSİYONLAR VE LİTERATÜRÜN GÖZDEN GEÇİRİLMESİ: ÜNİVERSİTE HASTANESİNDE YAPILAN RETROSPEKTİF BİR ÇALIŞMA

Sekonder enfeksiyonlar, COVID-19 ile hastaneye yatırılan hastalarda yüksek mortaliteye yol açan başlıca komplikasyonlar arasındadır. Bu çalışmada, COVID-19 hastalarında gelişen sekonder enfeksiyonların prevalansı, risk faktörleri, etiyolojik ajanları ve antimikrobiyal direnç paternlerinin belirlenmesi amaçlanmıştır. Çalışmaya 48 saat hastanede yattıktan sonra sekonder bakteriyel ve fungal enfeksiyon gelişen, COVID-19 PCR testi pozitif olan hastalar dahil edilmiştir. Hastaların klinik örneklerinden elde edilen bakteri ve mantar kültürlerinin sonuçları retrospektif olarak değerlendirilmiştir. Bu çalışmanın sonunda hastanede yatan 267 hastanın %16.1'inde (n=43) sekonder enfeksiyon geliştiği belirlenmiştir. Sekonder enfeksiyonlar erkeklerde (n=28, %65.1) kadınlara (n=15, %34.9) göre daha fazla saptanmıştır (p=0.024). Sekonder enfeksiyonu olan hastaların medyan yaşı (65.0 yıl) daha yüksek bulunmuştur (p<0.05). Sekonder enfeksiyonlu 43 hastanın %93'ünde (n=40) komorbidite saptanmıştır. Sekonder enfeksiyon gelişen hastaların 29'unun (%67.4) yoğun bakım ünitelerinde, 14'ünün (%32.6) servislerde tedavi gördüğü belirlenmiştir (p<0.001). Sekonder enfeksiyonların hastanede kalış süresini uzattığı (ortalama 25.5 gün) ve mortaliteyi artırdığı (n=16, %37.2) bulunmuştur (p<0.001). Etken ajan olarak tanımlanan ilk üç mikroorganizma, metisiline dirençli koagülaz negatif stafilokoklar (n=21, %16.3), Acinetobacter baumannii (n=19, %14.7) ve Candida albicans’tır (n=14, %10.9). Acinetobacter baumannii izolatlarının karbapenem direnç oranları %94.7 saptanmıştır. Koagülaz negatif stafilokokların %100'ünde metisilin direnci bulunurken vankomisin, teikoplanin ve linezolid direnci saptanmamıştır. C. albicans (%10.9) izolatlarında test edilen antifungal ajanlara karşı direnç bulunmamıştır. Sekonder enfeksiyonlar arasında özellikle pnömoni (n=25, %36.3), kan dolaşımı enfeksiyonları (n=19, %27.6) ve idrar yolu (n=18, %26.1) enfeksiyonları ilk sırada yer almıştır. COVID-19 ile ilişkili olarak gelişen sekonder enfeksiyonların ve risk faktörlerinin saptanması, etken mikroorganizmaların tanımlanması ve antimikrobiyal direnç paternlerinin belirlenmesi hastalığın prognozu, enfeksiyon kontrolü ve antimikrobiyal yönetimi açısından oldukça önemlidir

Time-Kill Kinetics and In Vitro Antifungal Susceptibility of Non-fumigatus Aspergillus Species Isolated from Patients with Ocular Mycoses

Aspergillus species can cause ocular morbidity and blindness, and thus, appropriate antifungal therapy is needed. We investigated the in vitro activity of itraconazole, voriconazole, posaconazole, caspofungin, anidulafungin, and amphotericin B against 14 Aspergillus isolates obtained from patients with ocular mycoses, using the CLSI reference broth microdilution methodology. In addition, time-kill assays were performed, exposing each isolate separately to 1-, 4-, and 16-fold concentrations above the minimum inhibitory concentration (MIC) of each antifungal agent. A sigmoid maximum-effect (Emax) model was used to fit the time-kill curve data. The drug effect was further evaluated by measuring an increase/decrease in the killing rate of the tested isolates. The MICs of amphotericin B, itraconazole, voriconazole, and posaconazole were 0.5–1.0, 1.0, 0.5–1.0, and 0.25 µg/ml for A. brasiliensis, A. niger, and A. tubingensis isolates, respectively, and 2.0–4.0, 0.5, 1.0 for A. flavus, and 0.12–0.25 µg/ml for A. nomius isolates, respectively. A. calidoustus had the highest MIC range for the azoles (4.0–16.0 µg/ml) among all isolates tested. The minimum effective concentrations of caspofungin and anidulafungin were ≤0.03–0.5 µg/ml and ≤0.03 µg/ml for all isolates, respectively. Posaconazole demonstrated maximal killing rates (Emax = 0.63 h−1, r2 = 0.71) against 14 ocular Aspergillus isolates, followed by amphotericin B (Emax = 0.39 h−1, r2 = 0.87), voriconazole (Emax = 0.35 h−1, r2 = 0.098), and itraconazole (Emax = 0.01 h−1, r2 = 0.98). Overall, the antifungal susceptibility of the non-fumigatusAspergillus isolates tested was species and antifungal agent dependent. Analysis of the kinetic growth assays, along with consideration of the killing rates, revealed that posaconazole was the most effective antifungal against all of the isolates